BURCKART, GJ

HASHEM, H

MAKOWKA, L

PTACHCINSKI, RJ

STARZL, TE

VENKATARAMANAN, R

WANG, CP

WONG, L

ZEEVI, A

D-Scholarship@Pitt

( ~~1F~F! /1D ,/9Z Cyclosporine Metabolites in Human Bile: Recovery and Immunologic Activity G.J. Burckart. C.P. Wang. A. Zeevi. R. Venkataramanan, R.J. Ptachcinski, H. Hashem. L. Wong. L. Makowka, and T.E. Starzl CYCLOSPORINE (CyA) is a highly metabolized cyclic peptide with potent immunosuppressive properties. Although the parent compound of Cy A is considered to be primarily responsible for the immunosuppres-sive activity, initial observations with purified metabolite obtained by synthesis or by recov-ery from animal studies suggests that metabo-lites M 17, M 1, and M21 have immunosup-pressant activity in vitro. I Human bile contains multiple CyA metabolites.2 and the contribution of its metabolites to the overall immunosuppressive effect of the drug is as yet unknown. The objective of this study was therefore to (1) recover multiple fractions from human bile and (2) test their inhibitory effect on the growth of human lymphocytes in vitro. MATERIALS AND METHODS Metabolite Recovery Pooled bile was collected from the T tubes of adult patients who had undergone orthotopic liver transplanta-tion. A fresh 50-mL aliquot of bile was extracted with ethyl ether in a separatory funnel, the ether layer was obtained, and ether was removed under reduced pressure. The residue was reconstituted with aqueous methanol and washed in a separatory funnel with n-hexane, and the hexane layer was discarded. The hexane wash was repeat-ed, and the aqueous methanol was taken to dryness under reduced pressure. The high-pressure liquid chromatographic (HPLC) system consisted of a gradient of acetonitrile:water pumped through a 15-cm, 5-/Lm C 18 column (Supelco, From the Clinical Pharmacokinetics and Immunopa-thology Laboratories. Schools of Pharmacy and Medi-cine. University of Pittsburgh. Address reprint requests to G.J. Burckart. PharmD. UniversityofPitrsburgh. 807 Salk Hall. Pittsburgh. PA 15261. © 1988 by Grune & Stratton. Inc. 0041-1345/88/2001-1071 $03.00/0 Bellefonte, PAl that was heated to 70°C. UV detection was performed at 214-nm wavelength. The complex gra-dient was adjusted to give the optimal separation of the component peaks over a period of I 18 minutes. The residue from the extraction was reconstituted with metha-nol, and the injection volume was adjusted to permit maximal injection with the best possible peak separation Component peaks were manually collected. the acetonl trile was removed, and the aqueous portion was lyophil' ized. Dry material from matched peaks of multiple injec-tions was combined, and the identity of the material was confirmed as previously reported. 2 Lymphocyte Proliferation Studies Human peripheral blood lymphocytes were isolated from heparinized blood by the Ficoll-Hypaque technique. The lymphocytes (l05/ well) were incubated at 37°C in 200 /LL tissue culture medium (TCM) supplemented with 10% human serum in the presence of concanavalin A (Con A, 10 /Lg/mL) for 72 hours. During the final 20 hours of incubation. each culture was labeled with I /LCi lH-thymidine. The cultures were harvested and counted in a liquid scintillation counter as previously described. Unidirectional human mixed lymphocyte reactlll (MLR) cultures were set up with 10' irradiated (2,000 R J stimulator cells in a volume of 200 /LL TCM supple-mented with 10% human serum for six days. Proliferation was assessed by the degree of lH-thymidine incorporation during the final 20 hours of incubation. The inhibitory effects of Cy A and bile-derived metabolite fractions on Con A and MLR reactivity were measured at concentra-tions ranging from 0.005 to 5.0 jlg/mL. The results are expressed as percent inhibition or (I - [cpm drug/cpm control]) x 100. RESULTS Thirteen major fractions were collected from the HPLC column, lyophilized, and combined with matched peaks to yield dry weights of between 0.65 and 2.41 mg. Figure i demonstrates an HPLC chromatogram of the injection of bile extract, the identification of each fraction on the chromatogram. and the corresponding identity of the peaks in relation to the nomenclature of Maurer.4 After identi-fication, further HPLC analysis demonstrated 190 Transplantation Proceedings, Vol XX. No 1. Suppl 1 (Februaryl, 1988: pp 190-192 1-CYCLOSPORINE METABOliTE IMMUNOlOGIC ACTIVITY Fig 1. An HPLC chromato-gram of an injection of bile extract. Collected fractions are shown as are corresponding eyA metabolite designations. .., l! c( '" a:o VJ '" c( that bile-derived MI, MI8. and M21 were >95o/r pure by peak areas. whereas bile-der; ',ed M 17 contained approximately 10% of an unidentified compound. The 13 compo-nents, including bile-derived M 17, M I, M 18. and M2I, were subjected to immunologic testing. In the test of inhibition with Con A-induced proliferation of peripheral blood lym-phocytes. greater than 80% inhibition was observed with CyA concentrations of 0.5 /-Lg/ mL or greater. Bile-derived M17. MI, and M21 produced approximately 60%.50%, and 30% inhibition at the highest concentration (5 ILg/mL). but no effect was noted in any of the~e fractions at 0.05 ILg/mL or lower. The san.c ievel of 50% inhibition of the Con A-stimulated response was observed with CyA in a IOO-fold lower concentration (0.05 ILg/mL). Figure 2 shows the results of the MLR stud-ies. Greater than 80% inhibition of the MLR was observed for Cy A at concentrations as low as 0.05 ILg/mL. Significant inhibition was i f~I:;! nell CE I"l.R 40 RESPOOSE 191 7 8 9 10 II 12 13 IMI7ICMI)CMI8) 1M2!) FRACTION NUMBER observed with fraction 4 (unidentified) and with bile-derived M13. M17. MI. and M21 at the highest (5.0 /-Lg/mL) concentration. Bile-derived M 17 exhibited inhibitory action in the MLR similar to CyA even after the tenfold and 1 ,ODD-fold dilution. DISCUSSION CyA metabolites can be recovered from human bile and do have pharmacological activity when tested agianst in vitro lympho-cyte cultures. Human bile contains many Cy A metabolites, a number of which are only now being identified. 5 Con A stimulation of peripheral blood lymphocytes was inhibited by several bile-derived metabolite fractions, with relative potency being CyA > M 17 > MI > M21 > MI8. Although five bile frac-tions produced 40% or greater inhibition of the MLR in the 5.0 J,Lg/mL concentration, the activity of M 17 was particularly striking in that it paralleled the activity of CyA even in 1 ,ODD-fold dilution. This observation is partic-0+------0----......::"-8'-----· Fig 2. Percent inhibition of an MLR v concentration of CyA. its metabolites. and a fraction ("4) from the HPLC collection. 5 .5 ,05 u05 Drug ug/ml 192 ularly important in that M 17 frequently is found in the blood of transplant patients in equal or greater concentrations than CyA. Although the exact contribution of the metab-BURCKART ET ;. olites to overall immunosuppressant activity is unclear, future studies of the action of CyA must take into account the presence of M 17 and other metabolites. REFERENCES I. Freed BM. Rosano TG. Lempert N: Transplanta-tion43:123,1987 2. Burckart GJ, Starzl TE. Venkataramanan R. et al: Transplant Proc 18:46, 1986 3. Zeevi A. Duquesnoy R: J Immunogenet 12:17. 1985 4. Maurer G: Transplant Proc 17: 19. 1985 5. Hashem H. Venkataramanan R. Burckart GJ. et al: Transplant Proc (this issue) 

CYCLOSPORINE METABOLITES IN HUMAN BILE - RECOVERY AND IMMUNOLOGICAL ACTIVITY

Burckart, GJ

Wang, CP

Zeevi, A

Venkataramanan, R

Ptachcinski, RJ

Hashem, H

Wong, L

Makowka, L

Starzl, TE

Name not available

Cyclosporine metabolites in human bile: Recovery and immunologic activity

http://d-scholarship.pitt.edu/4221/1/31735062129865.pdf

CYCLOSPORINE METABOLITES IN HUMAN BILE - RECOVERY AND IMMUNOLOGICAL ACTIVITY

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